Device for solid phase extraction and method for purifying samples prior to analysis

ABSTRACT

A solid phase extraction (SPE) device having a reservoir with an opening; a well comprising an internally tapered wall, the well having a wider interior diameter at an end closest to the opening than at an exit spout; a first filter within the well; a bed of sorbent particles within the well below the first filter; and a second filter having a diameter smaller than the first filter within the well below the bed of sorbent particles and above the exit spout is provided. A method of performing SPE using the device is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.10/100,762 filed Mar. 19, 2002, now pending. The contents of theaforementioned application are hereby expressly incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Solid phase extraction (SPE) is a chromatographic technique forpreparing samples prior to performing quantitative chemical analysis,for example, via high performance liquid chromatography (HPLC), or gaschromatography (GC). The goal of SPE is to isolate target analytes froma complex sample matrix containing unwanted interferences, which wouldhave a negative effect on the ability to perform quantitative analysis.The isolated target analytes are recovered in a solution that iscompatible with quantitative analysis. This final solution containingthe target compound can be directly used for analysis or evaporated andreconstituted in another solution of a lesser volume for the purpose offurther concentrating the target compound, making it more amenable todetection and measurement.

[0003] Depending on the type of analysis to be performed, and detectionmethod used, SPE may be tailored to remove specific interferences.Analysis of biological samples such as plasma and urine using highperformance liquid chromatography (HPLC) generally requires SPE prior toanalysis both to remove insoluble matter and soluble interferences, andalso to pre-concentrate target compounds for enhanced detectionsensitivity. Many sample matrices encountered in bio-separations containbuffers, salts, or surfactants, which can be particularly troublesomewhen mass spectrometer based detection is used. SPE can also be used toperform a simple fractionation of a sample based on differences in thechemical structure of the component parts, thereby reducing thecomplexity of the sample to be analyzed.

[0004] Devices designed for SPE typically include a chromatographicsorbent which allows the user to preferentially retain samplecomponents. Once a sample is loaded onto the sorbent, a series oftailored washing and elution fluids are passed through the device toseparate interferences from target sample components, and then tocollect the target sample components for further analysis. SPE devicesusually include a sample holding reservoir, a means for containing thesorbent, and a fluid conduit, or spout for directing the fluids exitingthe device into suitable collection containers. The SPE device may be ina single well format, which is convenient and cost effective forpreparing a small number of samples, or a multi-well format, which iswell suited for preparing large numbers of samples in parallel.Multi-well formats are commonly used with robotic fluid dispensingsystems. Typical multi-well formats include 48-, 96-, and 384-wellstandard plate formats. Fluids are usually forced through the SPE deviceand into the collection containers, either by drawing a vacuum acrossthe device with a specially designed vacuum manifold, or by usingcentrifugal or gravitational force. Centrifugal force is generated byplacing the SPE device, together with a suitable collection tray, into acentrifuge specifically designed for the intended purpose.

[0005] Various means have been used to contain chromatographic sorbentswithin SPE devices. A common method, described in U.S. Pat. No.4,211,658, utilizes two porous filters, with chromatographic sorbentcontained between the filters. In this design, the SPE device isessentially a small chromatographic column containing a packed bed ofsorbent. A variation of this design is described in U.S. Pat. No.5,395,521, where the porous filter elements are spherical in shape. InU.S. Pat. No. 4,810,381, the chromatographic sorbent is immobilizedwithin a thin porous membrane structure. In EP Application No. 1110610A1a method is described for securing these filters within the SPE deviceby means of a sealing ring pressed around the periphery of the membranedisc. In U.S. Pat. No. 5,486,410 a fibrous structure containingimmobilized functional materials is described. In U.S. Pat. No.5,906,796 an extraction plate is described where glass fiber discscontaining chromatographic sorbent are press fit into each well of theSPE device.

[0006] A number of chromatographic sorbents can be used depending on thenature of the sample matrix and target compounds. A common example is touse porous silica that has been surface derivatized with octydecyl (C₁₈)or octyl (C₈) functional groups. Porous particles that are based onorganic polymers are also widely used. One such type, which isparticularly well suited for SPE due to its high loading capacity andunique retention properties, is described in U.S. Pat. No. 6,254,780.

[0007] Typical SPE methods contain a sequence of steps, each with aspecific purpose. The first step, referred to as the “conditioning”step, prepares the device for receiving the sample. For reversed-phaseSPE, the conditioning step involves first flushing the SPE device withan organic solvent such as methanol or acetonitrile, which acts to wetthe surfaces of both the device and the sorbent, and also rinses anyresidual contaminants from the device. This initial rinse is generallyfollowed with a highly aqueous solvent rinse, often containing pHbuffers or other modifiers, which will prepare the chromatographicsorbent to preferentially retain the target sample components. Onceconditioned, the SPE device is ready to receive the sample.

[0008] The second step, referred to as the “loading” step, involvespassing the sample through the device. During loading, the samplecomponents, along with many interferences are adsorbed onto thechromatographic sorbent. Once loading is complete, a “washing” step isused to rinse away interfering sample components, while allowing thetarget compounds to remain retained on the sorbent. The washing step isthen followed by an “elution” step, which typically uses a fluidcontaining a high percentage of an organic solvent, such as methanol oracetonitrile. The elution solvent is chosen to effectively release thetarget compounds from the chromatographic sorbent, and into a suitablesample container.

[0009] In many cases, elution with high concentrations of organicsolvent requires that further steps be taken before analysis. In thecase of chromatographic analysis (HPLC), it is highly desirable forsamples to be dissolved in an aqueous-organic mixture rather than a pureorganic solvent, such as methanol or acetonitrile. For this reason, SPEsamples eluted in pure acetonitrile or methanol are usually evaporatedto dryness (“drydown”), and then reconstituted in a more aqueous mixture(“reconstitution”) before being injected into an HPLC system. Theseadditional steps not only take time and effort, but can also lead toloss of valuable sample, either through target analyte loss ontocollection container surfaces during drydown, or due to target analyteevaporative losses or difficulties encountered when trying tore-dissolve the dried sample in the higher percent aqueous fluid.

[0010] It can be seen then, that it is advantageous for an SPE device tohave a high capacity for retaining target compounds of a wide range ofchromatographic polarities, to be capable of maintaining target compoundretention as sample interferences are washed to waste, and then toprovide the capability to elute target compounds in as small an elutionvolume as possible, thereby maximizing the degree of target compoundconcentration obtained during SPE.

[0011] The ability to elute in very small volumes of solvent has theadded benefit of minimizing the amount of time required to evaporate andreconstitute the sample before proceeding with analysis if furtherconcentration or solvent exchange is required. If elution volume can bekept very low, then drydown and reconstitution can be entirelyeliminated.

[0012] Traditional SPE device designs have attempted to address theseissues, each with a limited measure of success. Packed bed devicesutilize packed beds of sorbent particles contained between porous filterdiscs that are press fit into the SPE device. The capture efficiency ofthe resulting packed beds is typically quite good, especially if thesorbent properties are carefully chosen. One drawback with conventionalpacked bed devices is that the void volume contained within the porousfilters and packed bed requires that relatively large elution volumes beused to completely elute the target compounds. Typical elution volumesrequired to fully elute target compounds from a packed bed type SPEdevice fall in the range of 200-5000 μL, depending on the size of thesorbent bed.

[0013] Membrane based designs attempt to address this issue by embeddingsorbent particles within a fluorocarbon based membrane, which are thenplaced into the SPE device. A small mass of sorbent particles isembedded into a thin membrane structure with a wide cross sectionalarea. Since the membrane does not require retaining filters, the volumeassociated with the two porous filters is eliminated. This approachreduces the total volume contained within the device, and therefore thevolume of solvent required for elution. A typical elution volumerequired to fully elute target compounds from a particle in membrane SPEdevice fall in the range of 75-500 μL. Designs of this type havedrawbacks in other areas however. The sorbent particles are less denselypacked within the membrane structure than within a packed bed, leadingto poorer capture efficiency, and a greater chance that target compoundswill break through the device without being well retained. In addition,the flow properties of the membrane can be highly variable, due to thepoor wetting characteristics of the fluorocarbon based membrane whenusing highly aqueous fluids.

[0014] In U.S. Pat. No. 5,906,796 a design is described in which glassfiber based extraction discs containing chromatographic particles arepress fit into each well of the SPE device. Like the membrane designs,this approach immobilizes the sorbent particles in a thin sheet, therebyminimizing device void volume and required elution volumes. Typicalvolumes required to fully elute target compounds from an SPE device suchas this fall in the range of 75-500 μL, which is comparable toparticle-in-membrane devices. The sorbent particles are even lessdensely packed than with membranes however, so sample breakthrough tendsto be higher than with either membrane or packed bed devices, andsorbent particles can often break free from the fibrous matrix andcontaminate the collected sample solution. One advantage over membranedevices is that flow problems due to wetting issues are generally lesscommon due to the more open structure of the glass fiber disc. Onedisadvantage of this particle embedded glass fiber disk is that itcontains silanol groups that interact with basic compounds. Thisrequires the use of more complex elution solvents, for example, theaddition of 2% base or acid to the elution solvent, to maintain the75-500 μL elution volumes.

[0015] It can be seen then, that the lower elution volume capabilityachieved with both the membrane and glass fiber approaches is at theexpense of target compound breakthrough during loading and/or poorrecovery for non-polar compounds. Although the volume of fluid needed toeffectively elute samples from the membrane and glass fiber formats isreduced to approximately one half of the volume required withconventional packed bed based devices, dry-down and reconstitution stepsare still required before samples can be further analyzed by HPLC.

SUMMARY OF THE INVENTION

[0016] The present invention relates to an improved SPE device which hasbeen specifically designed to contain a small packed bed ofchromatographic sorbent such that the bed provides for highly efficientretention of target compounds, while the volume contained within thesorbent bed is sufficiently small as to allow for efficient elution ofsample compounds in a minimal elution volume. Specifically, the solidphase extraction device of the present invention comprises a reservoirwith an opening; a well comprising an internally tapered wall, the wellhaving a wider interior diameter at an end closest to the opening thanat an exit spout; a first filter within the well; a bed of sorbentparticles within the well below the first filter; and a second filterhaving a smaller diameter than the first filter within the well belowthe bed of sorbent particles and above the exit spout.

[0017] The present invention provides a large bed height to top beddiameter ratio using a significantly smaller sorbent mass than ispresent in current state of the art devices. The large bed height to beddiameter ratio enhances the retention of target compounds and helps toprevent breakthrough of these compounds during the load and wash steps.In SPE the first filter and the top of the sorbent bed acts like a depthfilter in removing insoluble sample components. The larger diameter forthe upper portion of the bed and larger diameter first filter allows thedevice to draw through larger sample volumes than could be drawn througha device having an upper bed diameter the same as the lower bed diameterbefore obstructions will occur. The smaller second filter increases thebed height to bed diameter ratio for a given mass of sorbent whilereducing the hold up volume of the device which minimizes requiredelution volumes.

[0018] Moreover, the present invention provides for conically shapedpacked beds contained between spherical filters which enhance theperformance of solid phase extraction devices by allowing targetcompounds to be both efficiently retained and eluted. The larger firstspherical filter provides a surface area that is approximately two timesthe area of an equivalently sized cylindrical filter. For example,surface area of the top half of a sphere (π/2×d²) of a diameter of 0.1″is equal to the surface area of the top of a disk of diameter 0.14″(π/4×d²). The smaller second filter helps to minimize the amount ofsorbent needed to create a bed length that will be free of adverseimperfections.

[0019] The present invention enables the retention of target compoundswith a wide range of chromatographic polarity with elution in volumesthat are much reduced from the current state of the art for solid phaseextraction. This reduction in elution volume provides a solutioncontaining the target compounds that can be diluted with an aqueoussolution while still maintaining the high sample concentrations requiredfor analysis.

[0020] According to another aspect, the present invention provides anenhanced method of performing solid phase extraction, where the volumeof elution solvent is sufficiently small so as to eliminate the need foran evaporation step. The method involves elution of the target compoundsin a minimal volume of organic solvent, typically 10-40 μL, which isthen diluted with a highly aqueous fluid to form an aqueous organicsample mixture. This mixture is suitable for direct analysis by HPLC,thereby eliminating the time, expense, and potential sample lossesassociated with evaporation and reconstitution steps, while stillmaintaining a high degree of target compound(s) concentration.

[0021] Specifically, the inventive method comprises the steps ofproviding the above-mentioned SPE device, and isolating targetsubstances from interfering components in a sample medium, wherein thetarget substances are substantially eluted in less than 50 μL volume.

[0022] In one embodiment of the present invention, the isolating step ofthe present invention preferably includes conditioning the SPE devicewith an organic solvent; equilibrating the SPE device with an aqueoussolution; adding a prepared sample containing the target substances andinterfering components to the SPE device; washing the SPE device with anaqueous solution to remove interfering components; and eluting theadsorbed target substances.

[0023] In a preferred embodiment of this enhanced method of the presentinvention, the aqueous diluent is added directly through the SPE device,while still on the processing station used to perform the SPE fluidtransfers. In this way, residual elution solvent is swept through thedevice into the collection container, where it is diluted by the aqueousfluid and mixed by the gentle air stream that is drawn through the wellat the end of the transfer. This approach has the advantage ofeliminating the need for a separate pipetting operation to perform thedilution step.

SUMMARY OF THE DRAWINGS

[0024]FIG. 1 is an illustration of a single well embodiment of thepresent invention, where the internal well geometry is a simple taperedshape, containing two spherical filter elements of different sizes, abed of sorbent particles contained between the filters, an exit spoutdownstream from the smaller filter, and a fluid reservoir upstream fromthe larger porous filter.

[0025]FIG. 2 is an illustration of a single well embodiment of thepresent invention where the internal tapered well geometry is segmented,providing an exit spout downstream from the smaller porous filter, alower tapered section containing both the smaller porous filter and abed of sorbent particles, an upper tapered section which contains thelarger porous filter upstream from the sorbent bed, and a transitionsection leading to an upper fluid reservoir.

[0026]FIG. 3 is an illustration of a multi-well SPE device where eachwell of the device contains the single well device geometry of FIG. 2.

[0027]FIG. 4 is a graph illustrating the effect of hold-up volume onrecovery in 25 μL elution volumes.

[0028]FIG. 5 is an illustration of a trapezoidal exit spout.

[0029]FIG. 6 is an illustration of a semi-circular exit spout of thedevice.

DETAILED DESCRIPTION OF THE INVENTION

[0030]FIG. 1 shows an SPE device of packed bed design where the devicehas been optimized for high capture efficiency, while requiring minimalelution volume. The device (11) contains a small bed of sorbentparticles (12), contained between two porous filter elements ofdifferent sizes (13 & 14) within a tapered internal well geometry (15),such that the porous filter (13) positioned downstream from the sorbentbed (12) is smaller than the porous filter (14) positioned upstream fromthe sorbent bed. The device also includes a reservoir section (16)located upstream from the larger porous filter (14) and an exit spout(17) located downstream from the smaller porous filter (13). The spoutdirects fluids exiting the device into a suitable collection container(not shown).

[0031] Porous filters (13 & 14) may be of any material suitable forretaining the sorbent particles. In a preferred embodiment, porousfilters (13 & 14) are made from sintered polyethylene material. FIG. 1represents a single well version of the device, although it will beobvious to one skilled in the art, that the device may also beconfigured as part of a multi-well SPE device.

[0032] In this preferred embodiment of the SPE device of the presentinvention, the porous filters that contain the sorbent particles arespherical in shape, and the sorbent bed is configured in a taperedgeometry, with the downstream porous spherical filter being smaller thanthe upstream porous spherical filter.

[0033] The sorbent particles employed in the device include anyparticulate matter that is capable of having at least one substance,either target or interfering, adhered thereto. Illustrative examples ofsorbent particles that may be employed in the present invention include,but are not limited to: ion exchange sorbents, reversed phase sorbents,and normal phase sorbents. More particularly, the sorbent particles maybe an inorganic material such as SiO₂ or an organic polymeric materialsuch as poly(divinylbenzene). In some embodiments of the presentinvention, the sorbent particles may be treated with an organicfunctional group such as a C₂-C₂₂, preferably C₈-C₁₈ functional group.One skilled in the art will find it obvious that the size, shape,surface area, and pore volume of the sorbent particles, may all bemodified to suit specific applications without departing from the scopeof the invention.

[0034] The tapered internal device geometry acts to provide an upstreamfirst porous filter having a large filtration area for capturing foreignsample particulates prior to them reaching the sorbent bed, and asmaller downstream filter, while allowing minimal internal void volumebetween the sorbent bed and the first filter. The effective filtrationarea of the spherical filter is based on the surface of the exposedhemispherical section of the filter, which is larger than the area of aflat disc filter of equal diameter by a factor of 2.

[0035] The spherical filters are easy to handle during assembly andrequire no special insertion tooling. Moreover, the spherical filtersself-align when placed into a tapered well cavity, and seal against thecavity wall easily without the need for close dimensional tolerancesbetween the spherical filters and the internal surface of the well. Thetapered well design also allows for a range of sorbent masses within thesame SPE device, thereby providing flexibility to tailor the device fordifferent applications. This is accomplished by simply changing thediameter of the spherical porous filters, thereby positioning thefilters and packed sorbent bed either higher or lower within the tapereddevice without having to alter the well cavity.

[0036] The tapered well geometry differs from conventional cylindricaldesigns, since it results in a sorbent bed shape that has considerablyless tendency to form undesirable flow channels, thereby preventingsample components bypassing the bed without adequately contacting thesorbent particles. Fluids passing through the sorbent bed during theconditioning and loading steps act to consolidate the tapered packedbed, resulting in a consistently formed bed structure. This results inefficient contact between the sample and the sorbent bed, less chancefor sample breakthrough during loading, and efficient use of wash andelution fluids.

[0037]FIG. 2 is an illustration of a single well embodiment of thepresent invention where the internal tapered well geometry is segmented,providing an exit spout (17) downstream from the smaller second porousfilter (13), a lower tapered section (18) containing both the smallerporous filter (13) and a bed of sorbent particles (12), an upper taperedsection (19) which contains the larger first porous filter (14) upstreamfrom the sorbent bed (12), and a transition section (20) leading to anupper fluid reservoir (21) having a larger diameter opening (22).Segmentation of the internal taper in this way allows for SPE deviceswhich have larger capacity reservoirs while maintaining the advantagesof the present invention in a relatively short overall well height.

[0038]FIG. 3 is an illustration of a multi-well version of an SPE deviceincorporating the single well design of FIG. 2. Common multi-wellformats include plate designs based on the common 48, 96, and 384standard well formats.

[0039] The exit spout directs fluids into any suitable container. In thepreferred embodiment, shown in FIG. 5, the exit spout 17 geometry issubstantially trapezoidal. This geometry is used to prevent the exitingfluids from creeping up the exterior wall of the device and provideseffective beading and dropping of the exiting fluids. A semi-circularshape may also be used for the exit spout 17 as shown in FIG. 6.

[0040] The present invention can be used to purify samples prior toanalysis, i.e., to isolate a desired target substance from aninterfering substance in a sample medium, using a smaller elution volumethan heretofore possible with prior art SPE devices. Specifically, andin a preferred embodiment, the method of the present invention comprisesfirst conditioning the SPE device with any organic solvent that iscapable of wetting the surfaces of the device and sorbent particles.Illustrative examples of organic solvents that can be used in theconditioning step include, but are not limited to: polar or non-polarorganic solvents such as methanol and acetonitrile. The amount oforganic solvent used to condition the SPE device may vary and is notcritical to the present invention so long as the organic solvent is usedin an amount that wets the SPE device. Note that the solvent used inthis step of the inventive method also serves to remove contaminantsfrom the SPE device.

[0041] After the conditioning step, an aqueous solution is used toequilibrate the conditioned SPE device. The amount of aqueous solutionused to equilibrate the SPE device may vary and is not critical to thepresent invention.

[0042] A prepared sample containing at least one target substance aswell as interfering components is then added to the SPE device usingconventional means that are well known to those skilled in the art. Theinventive method is not limited to a specific prepared sample or targetsubstance. For example, the prepared sample may be blood plasma, serum,urine, and other like samples that are capable of being purified bysolid phase extraction. Insofar as the target substance is concerned,the inventive SPE method works well on polar compounds, non-polarcompounds, acidic compounds, neutral compounds, basic compounds and anymixtures thereof.

[0043] Next, an aqueous solution is employed to remove the interferingsubstance from the SPE device and thereafter the target substance, whichis adsorbed onto the sorbent particles, is eluted from the SPE deviceusing an organic eluant that is capable of removing the adsorbed targetsubstances from the SPE device.

[0044] The following examples are given to illustrate the scope of thepresent invention. Because these examples are given for illustrativepurposes only, the present invention is not limited to the followingexamples.

EXAMPLES Example 1

[0045] A spherical porous polyethylene filter having a diameter of0.075″ is press sealed into a molded well cavity having a 5° includedangle taper as shown in FIG. 2. A packed bed is formed within the 5°tapered well using 2 milligrams of Waters' Oasis® HLB, 30 micron sorbentparticles. A spherical porous polyethylene filter having a diameter of0.100″ is press sealed into the upper section of the well which containsa 1° included angle. The upper porous filter acts to both contain thesorbent particles within the well, and to act as a sample pre-filter.

Example 2

[0046] A spherical porous polyethylene filter having a diameter of0.058″ is press sealed into a well having a 5° included angle taper asin EXAMPLE 1. A packed bed is formed within the 5° tapered well using 1milligram of Waters' Oasis® HLB brand, 30 micron sorbent particles. Aspherical porous polyethylene filter having a diameter of 0.100″ ispress sealed into the upper section of the well to both contain thesorbent particles within the well, and to act as a sample pre-filter.The resulting device contains one half the amount of sorbent as inEXAMPLE 1, but due to the smaller lower filter size, the bed ispositioned lower in the tapered well, with a bed shape that is wellsuited for effective performance.

Example 3

[0047] The SPE device of EXAMPLE 1 is placed on a vacuum manifoldstation with a collection vial positioned below the exit spout tocollect fluids exiting the device. A vacuum of 10″ Hg is applied to drawfluids through the device. The device is first conditioned by passing100 μL methanol through the device, followed by 100 μL water. A spikedplasma sample is prepared by spiking 250 μL porcine plasma with 1.9 μgof amitriptyline, followed by dilution with 250 μL of 2% phosphoric acidin water. The diluted spiked plasma sample is then drawn through thedevice. After addition of the diluted, spiked plasma sample, the sorbentbed is washed using 100 μL water. An elution step is then performed bypassing 25 μL acetonitrile/methanol (80/20 by volume) through thesorbent bed, and collecting into a clean collection vial. The resultingsample mixture contains the target compound, free from plasmainterferences, concentrated ten fold. The sample solution may beanalyzed directly, or further evaporated and reconstituted in a solventmixture suitable for the intended analysis.

Example 4

[0048] The SPE device of EXAMPLE 1 is used in identical manner asdescribed in Example 3, except that after eluting with 25 μLacetonitrile/methanol (80/20 by volume), an additional 25 μL water isdrawn through the sorbent bed and into the same vial which contains thepreviously eluted sample compound. The resulting sample mixture containsthe target compound, free from plasma interferences, concentrated fivefold in a 50% aqueous/organic solution, which is well suited for directanalysis using HPLC.

Example 5

[0049] The model target compounds acetaminophen, N-acetyl-procainamide,betamethasone, caffeine, naproxen, amitriptyline, and propranolol wereobtained from Sigma Aldrich. The model target compound practolol waspurchased from Tocris. The Octadecyl (C₁₈) SD-C18 3M Empore™ HighPerformance Extraction Disk Plate (PN 6015) was purchased from FisherScientific. The Universal Resin (UR) 3M Empore™ High PerformanceExtraction Disk Plate (PN 6345) was purchased from VWR. The Ansys®Technologies, INC. Spec·C18 96-Well Plate (PN 596-03) was purchased fromAnsys® Technologies, INC. The 5 mg Oasis® HLB Extraction Plate waspurchased from Waters (PN 186000309). A 2 mg amount of Oasis® HLB(Waters Corporation) was packed into a device similar to that shown inFIG. 1 with the sorbent contained between a lower polyethylene sphericalfrit of a diameter of 0.08″ at the outlet and an upper polyethylenespherical frit of a diameter of 0.1″ at the inlet. Organic solvents wereobtained from VWR (J. T. Baker HPLC grade).

[0050] Stock 1 mg/mL solutions of each of the following model targetcompounds were made in 20/80 methanol/water (v/v): acetaminophen,practolol, N-acetyl procainamide, caffeine, propranolol, andamitriptyline. Stock 1 mg/mL solutions of each of the following modeltarget compounds were made in 80/20 methanol/water (v/v): naproxen,betamethasone, and ibuprofen. The internal standard solution wasprepared by adding equal parts of the ibuprofen stock solution to water(1:1). Appropriate amounts of the stock solutions were added to a pH 7isotonic saline solution to achieve the following concentration of modeltarget compounds: Concentration Compound In Saline Test Mix practolol 5μg/mL n-acetyl procainamide 7.5 μg/mL acetaminophen 5 μg/mL caffeine 7.5μg/mL naproxen 5 μg/mL Amitriptyline 7.5 μg/mL betamethasone 2.5 μg/mLpropranolol 40 μg/mL Phenyl acetic acid 150 μg/mL

[0051] All solid phase extraction devices were conditioned with 100 μLof methanol, followed by 100 μL of water. Care was taken not to allowthe sorbent to dry out between the methanol and water rinse steps. 100μL of the saline solution containing the target model compounds wasdrawn through the device typically using <4″Hg vacuum. 100 μL of waterwas drawn through the device to wash the sorbent. 25 μL or 75 μL of an80/20 acetonitrile/methanol solution was drawn through the device toelute the model target compounds. 50 μL of a 0.5 mg/mL ibuprofeninternal standard solution and an additional 25 μL of saline was addedto each sample prior to analysis. Samples were analyzed by HPLC usingthe following gradient of 0.01% formic acid (D) to acetonitrile (C):Time Flow % A % B % C % D Curve 2.00 0.0 0.0 0.0 100.0 7.33 2.00 0.0 0.065.0 35.0 6 8.60 3.00 0.0 0.0 100.0 0.0 1 8.84 4.00 0.0 0.0 100.0 0.0 19.00 2.00 0.0 0.0 0.0 100.0 1 9.50 3.00 0.0 0.0 0.0 100.0 1 15.00 2.000.0 0.0 0.0 100.0 6 35.00 2.00 0.0 0.0 100.0 0.0 11 45.00 0.00 0.0 0.0100.0 0.0 11

[0052] The column temperature was maintained at 30° C. using a SparkHolland Mistral heater box. The HPLC system consisted of a Waters 600ESolvent Delivery System, a Waters 717plus Autosampler, a Waters in-linevacuum degasser, and a Waters 2487 Tunable UV detector set to 254 nm(sampling rate=2 points/sec). Millennium³² Chromatography Manager v3.20was used for data acquisition and processing, and equipment control.

[0053] Separations were performed using a 3.5 μm Symmetry Shield RP84.6×75 mm (Waters part number Wat094263) column with a 5 μm SymmetryShield RP8 Sentry 3.9×20 mm guard column (Waters Part Number Wat200675).The injection volume was 10 μL for all standards, controls, and samples.The total run time was 15 min.

[0054] The hold-up volume was determined for each of the devices tested.It was determined by adding 50 μL or 75 μL, depending on estimates ofthe device's hold-up volume, of 50/50 isopropanol/water to 4 wells each.The solution was allowed to soak into the beds for 30 sec. A vacuum offirst 4″Hg for 45 sec then 7″Hg for 45 sec was used to draw the solutionthrough the devices and into total recovery vials (Waters PN186000837).The volume of solution in the vials was measured using an auto-pipette.The hold-up volume was determined by subtracting the collected volumefrom the added volume.

[0055] The recovery results in Table 1 show the performance differencebetween what is commercially available on the market today and this newtip design. The data shows that recoveries in 25 μL volumes ranged from84% to 97% on the new tip device compared to 51%-86% on the bestperforming commercially available device today, which also contains thesame sorbent. This direct comparison illustrates how the new deviceformat improves recoveries. Devices containing particles embedded inglass fibers or Teflon had recoveries that were substantially lower (0to 64%).

[0056] The target compounds are listed in Table 1 from the most polar atthe top of the list to the least polar. On the Oasis® HLB 5 mg 96-wellplate, the recovery results sharply decrease as the polarity of thecompounds decreases. The new tip device is able to give high recoveriesfor compounds having a wide range of chromatographic polarities. TABLE 1% Recoveries (n = 4) in 25 μL Elution Volumes with 80/20Acetonitrile/Methanol Inventive Ansys ® 3M Device† Oasis ® Spec PlusEmpore ™ Oasis ® HLB 5 mg C₁₈ Plate Universal HLB 2 mg Plate glass Resinpacked bed packed bed fiber disk teflon disk Elution Volume 25 μL 25 μL25 μL 25 μL N-acetyl 95.6 85.8 0 6.9 procainamide practolol 92.8 83.6 07.7 acetaminophen 93.1 77.5 50.4* 9.1 caffeine 97.0 82.3 41.3 9.8propranolol 89.3 70.2 0 1.8 amitriptyline 83.6 59.1 0 0 betamethasone91.5 59.9 24.3 0.7 naproxen 84.1 50.8 64.4 5.3 Max Recovery 97 86 64 10Min Recovery 84 51 0 0

[0057] All others show <5% breakthrough.

[0058] The relative standard deviations (% RSDs) for the recoveries areshown in Table 2. They range from 0.9%-4% on the new tip design versus4.6%-10.5% on the best performing current state of the art device.Results with equivalent recoveries and reproducibilities to thoseobtained on the new tip design were not obtained on the existing 96-wellplates with less than 75 μL elutions. For all quantitative analyticalwork good reproducibility is essential and high recovery is desirable.For high sensitivity quantitative analytical work both are essential:good reproducibility and high recovery. TABLE 2 % RSDs (n = 4) forRecoveries in 25 μL Elution Volumes with 80/20 Acetonitrile/MethanolInventive Ansys ® 3M Device† Oasis ® Spec Plus Empore ™ Oasis ® HLB 5 mgC₁₈ Plate Universal HLB 2 mg Plate glass Resin packed bed packed bedfiber disk teflon disk Elution Volume 25 μL 25 μL 25 μL 25 μL N-acetyl0.9 5.0 72.9 procainamide Practolol 1.2 6.1 69.1 Acetaminophen 1.6 6.410.0 59.2 Caffeine 1.7 4.6 8.6 58.0 Propranolol 1.6 6.7 71.3Amitriptyline 4.0 7.6 — Betamethasone 2.5 8.6 13.5 200.0 Naproxen 2.510.5 3.2 73.0 Max RSD 4.0 10.5 13.5 200.0 Min RSD 0.9 4.6 3.2 58.0

[0059] Table 3 shows the recovery results obtained using a 75 μL elutionvolume on commercially available 96 well SPE plates that have beenspecifically designed to minimize elution volume. The shortcoming of theOasis HLB 5 mg plate is that the recoveries vary with the polarity ofthe compounds due to insufficient elution volume. The shortcomings ofthe Ansys® device are two fold. First recoveries of the basic compoundsare extremely poor due to secondary interactions with the sorbent andglass fiber. The addition of about 2% acetic acid or 2% ammoniumhydroxide to the elution solvent would improve recoveries. Themanufacturer of this device recommends using 500 μL or less to elutecompounds from this device.

[0060] Neutral model compounds like caffeine, a polar compound, andbetamethasone, a non-polar compound, do not suffer from this problem.The 78.9% recovery for caffeine, and 67.6% recovery for betamethasoneshow that 75 μL is not an adequate elution volume to recover a broadpolarity range of compounds from the Ansys plate.

[0061] The 3M Empore™ devices also show recovery problems for the bases.In addition, the 51% and 56% recoveries for betamethasone show that 75μL elution volumes are not adequate to elute a broad polarity range ofcompounds from these devices. All four of these devices also suffer frombreakthrough of acetaminophen, a polar neutral compound. TABLE 3 %Recovery (n = 4) in 75 μL Elution Volumes with 80/20Acetonitrile/Methanol for Different 96-well Formats 3M Ansys ® Empore ™3M Oasis ® Spec Universal Empore ™ HLB Plus C18 Resin C18-SD plate 5 mgplate glass plate plate packed bed fiber disk Teflon disk Teflon diskElution Volume 75 μL 75 μL 75 μL 75 μL N-acetyl 85.2 7.0 57.4 73.3procainamide Practolol 81.7 19.8 56.0 82.3** acetaminophen 86.3** 68.2*71.5** 76.6* Caffeine 93.1 78.9 75.1 92.9 Propranolol 83.8 1.8 33.5 41.5Amitriptyline 85.3 0.6 20.1 6.1 betamethasone 87.8 67.6 51.1 55.6Naproxen 79.9 85.0 62.8 70.8 Max Recovery 93.1 85.0 75.1 92.9 MinRecovery 79.9 0.6 20.1 6.1

[0062] All others show <5% breakthrough. TABLE 4 % RSDs for Recoveries(n = 4) in 75 μL Elution Volumes with 80/20 Acetonitrile/Methanol forDifferent 96-well Formats 3M Ansys ® Empore ™ 3M Spec Universal Empore ™Oasis ® Plus C18 Resin C18-SD HLB plate glass plate plate plate 5 mgfiber disk teflon disk teflon disk Elution Volume 75 μL 75 μL 75 μL 75μL N-acetyl 9.7 21.3 9.1 5.5 procainamide Practolol 10.6 24.0 8.8 3.6acetaminophen 5.9 4.5 8.5 10.8 Caffeine 3.9 9.5 8.6 3.1 Propranolol 6.9138.8 4.3 19.5 Amitriptyline 3.8 200.0 13.8 47.5 betamethasone 1.3 16.75.5 35.0 Naproxen 3.0 3.1 7.7 19.1 Max RSD 10.6 200.0 13.8 47.5 Min RSD1.3 3.1 4.3 3.1

[0063] The hold-up volume for each of the devices tested was measuredand is shown in Table 5 along with the recoveries for three modelcompounds. The recoveries for these model compounds are highest for thenew tip device due to its low hold-up volume. The recoveries in Table 5show a trend of lower recoveries for devices with higher hold-up volumesas illustrated in FIG. 4. In FIG. 4, % recovery in 25 μL is plottedagainst the device hold-up volumes (V) in μL. Diamonds indicate caffeinedata, squares indicate betamethasone data and triangles indicatenaproxen data TABLE 5 The Effect of Hold-up Volume on Recovery in 25 μLElution Volumes Inventive Ansys ® 3M Device Oasis ® Spec Empore ™Oasis ® HLB 5 mg Plus C₁₈ Universal HLB 2 mg Plate Plate glass ResisnPlate packed bed packed bed fiber disk teflon disk Elution Volume 25.0μL 25.0 μL 25.0 μL 25.0 μL caffeine 97.0 82.3 41.3 9.8 betamethasone91.5 59.9 24.3 0.7 naproxen 84.1 50.8 64.4 5.3 Device hold up 16.0 28.036.0 64.0 volume (μL)

[0064] Packed beds having a bed height to top diameter ratio of <0.23are not able to efficiently retain or elute compounds due toimperfections in the packed bed. Simply going to a 2 mg amount ofsorbent in the existing Oasis® HLB plate will not provide a resultcomparable to those obtained on the new device containing 2 mg. This isillustrated with the data in Table 6 showing lower recoveries for allbut the most non-polar compounds on the plate containing 2 mg of sorbentcompared to the plate containing 5 mg of sorbent. Table 7 shows that theRSDs are worse on the 2 mg plate compared to the 5 mg plate. TABLE 6Effect of Bed Height to Top Diameter Ratio on Recovery in 25 μLInventive Device Oasis ® Oasis ® Oasis ® HLB 2 mg HLB 5 mg HLB 2 mgPlate Plate packed bed packed bed packed bed Elution Volume 25 μL 25 μL25 μL N-acetyl procainamide 95.6 53.3* 85.8 practolol 92.8 48.9* 83.6acetaminophen 93.1 49.5* 77.5 caffeine 97.0 59.4* 82.3 propranolol 89.362.2* 70.2 amitriptyline 83.6 60.3* 59.1 betamethasone 91.5 66.5* 59.9naproxen 84.1 53.7* 50.8 Bed height to top 0.97 0.092 0.23 diameterratio

[0065] All others show <5% breakthrough. TABLE 7 Effect of Bed Height toTop Diameter on Recovery RSDs in 25 μL Inventive Device Oasis ® Oasis ®Oasis ® HLB 2 mg HLB 5 mg HLB 2 mg Plate Plate packed bed packed bedpacked bed Elution Volume 25 μL 25 μL 25 μL N-acetyl 0.9 44.6 5.0procainamide Practolol 1.2 46.5 6.1 acetaminophen 1.6 42.0 6.4 Caffeine1.7 38.0 4.6 propranolol 1.6 29.2 6.7 amitriptyline 4.0 22.7 7.6betamethasone 2.5 17.5 8.6 Naproxen 2.5 20.5 10.5 Bed height to top 0.970.092 0.23 diameter

Example 6

[0066] Devices similar to those shown in FIG. 1 were manually packedusing 1.0±0.05 mg of 30 μm Oasis® HLB (Waters Corporation) containedbetween two polyethylene spherical frits: a 0.035″ spherical frit at thebottom of the bed and a 0.055″ spherical frit at the top of the bed.Sodium chloride, Angiotensin II, and p-toluamide were obtained fromSigma-Aldrich. Triethylamine (TEA), glacial acetic acid, trifluoroaceticacid (TFA), and HPLC grade acetonitrile were obtained from J. T. Baker.The 15-mer oligodeoxythymidine (15-mer oligo T) was obtained fromMidland Certified Reagent Company (Midland Tex.). The 0.1 Mtriethylammonium acetate (TEAAc) was prepared by adding 2.21 mL ofglacial acid and 5.58 mL of triethylamine to 350 mL of H₂O. The solutionwas mixed, adjusted to a volume of 400 mL and pH adjusted to pH 7 usingacetic acid. The 0.24% TFA, and 50% acetronitrile were prepared byvolume. The 50 mM NaCl was prepared by adding 0.0584 grams of NaCl to 1liter of H2O. The 0.1 M TEAAc with 50 mM NaCl was prepared by adding2.21 mL of glacial acid and 5.58 mL of triethylamine to 350 mL of 50 mMNaCl. The solution was mixed, adjusted to a volume of 400 mL with 50 mMNaCl and pH adjusted to pH 7 using acetic acid. The 60 μL DNA loadsample contained 1 μg of 15-mer oligo T and 1 μg of p-toluamide in the0.1 M TEAAc buffer with 50 mM NaCl. The 60 μL peptide load samplecontained 1 μg of Angiotensin II and 1 μg of p-toluamide in the 0.24%TFA. All solutions were drawn through the tips using a vacuum of <5″Hg.

[0067] DNA Desalting Method:

[0068] 1. Condition each tip (n=3) with 60 μL of acetonitrile followedby 60 μL of 0.1 M TEAAc buffer

[0069] 2. Load 60 μL/tip of the DNA sample

[0070] 3. Wash with 60 μL/tip of the 0.1 M TEAAc buffer followed by 60μL/tip of H₂O

[0071] 4. Elute each tip with 10 μL of 50% acetonitrile in H₂O

[0072] Peptide Method:

[0073] 1. Condition each tip (n=4) with 60 μL of acetonitrile followedby 60 μL of 0.24% TFA

[0074] 2. Load 60 μL/tip of the peptide sample

[0075] 3. Wash with 20 μL of the 0.24% TFA followed by 20 μL of H₂O

[0076] 4. Elute each tip with 10 μL of 50% acetonitrile in H₂O

[0077] The DNA desalting and peptide recovery results are presented inTable 8. The results in Table 8 show that excellent recoveries for smallmolecules (ie p-toluamide), biopolymers (15-mer oligo T) and peptidescan be obtained in 10 μL elution volumes. TABLE 8 Recoveries and RSDsfor 15-mer oligo T, Angiotensin II, and p-Toluamide % Recovery % RSD DNAMethod 15-mer oligo T 88.2 2.3 p-Toluamide 93.3 4.8 Peptide MethodAngiotensin II 101.6 1.2 p-Toluamide 96.7 4.7

[0078] According, it should be readily appreciated that the device andmethod of the present invention has many practical applications.Additionally, although the preferred embodiments have been illustratedand described, it will be obvious to those skilled in the art thatvarious modifications can be made without departing from the spirit andscope of this invention. Such modifications are to be considered asincluded in the following claims.

What is claimed:
 1. A method of providing for a range of sorbent masseswithin a solid phase extraction device comprising: providing an solidphase extraction device comprising a reservoir with an opening forreceiving fluids; a well comprising an internally tapered wall, the wellhaving a wider interior diameter at a first end closest to the reservoirthan at a second end close to an exit spout, the well for conducting anextraction; an exit spout at the second end of the well for dischargingfluids; a first filter press sealed between the internally tapered wallsof the well for retaining insoluble components of the fluids; a secondfilter having a smaller diameter than the first filter press-sealedbetween the internally tapered walls of the well spaced apart and towardthe exit spout from the first filter; a quantity of sorbent particlespartial filing the volume in the well between the first and secondfilters; and a void volume between the quantity of sorbent particles andthe first filter for separating the quantity of sorbent particles fromthe first filter; and adjusting the filter diameters whereby the filterssettle in different positions within the tapered well.
 2. The method ofproviding for a range of sorbent masses within a solid phase extractiondevice as in claim 1 further comprising: the reservoir and well in amulti-well array.
 3. A method of separating a target substance frominterfering components in a sample medium comprising: providing an solidphase extraction device (SPE) comprising a reservoir with an opening forreceiving fluids; a well comprising an internally tapered wall, the wellhaving a wider interior diameter at a first end closest to the reservoirthan at a second end close to an exit spout, the well for conducting anextraction; an exit spout at the second end of the well for dischargingfluids; a first filter press sealed between the internally tapered wallsof the well for retaining insoluble components of the fluids; a secondfilter having a smaller diameter than the first filter press-sealedbetween the internally tapered walls of the well spaced apart and towardthe exit spout from the first filter; a quantity of sorbent particlespartial filing the volume in the well between the first and secondfilters; and a void volume between the quantity of sorbent particles andthe first filter for separating the quantity of sorbent particles fromthe first filter; and substantially isolating a target substance usingthe solid phase extraction device.
 4. The method according to claim 3wherein: the mass of sorbent particles is less than 5 milligrams.
 5. Themethod according to claim 3 wherein the isolating step comprises thesteps of: conditioning the SPE device with an organic solvent;equilibrating the SPE device with an aqueous solution; adding a preparedsample containing the target substances and interfering components tothe SPE device; washing the SPE device with an aqueous-organic solutionto remove interfering components; and eluting the adsorbed targetsubstances.
 6. The method according to claim 5 wherein: the targetsubstance is substantially eluted in less than 50 μL volume.
 7. Themethod according to claim 5 further comprising: diluting the elutedtarget substances by passing a diluent through the solid phaseextraction device.
 8. The method according to claim 3 where the samplemedium is blood plasma, urine or serum.
 9. The method according to ofclaim 3 where the target substance comprises at least one polarcompound, non-polar compound, acidic compound, neutral compound,biopolymer, basic compounds, and mixtures thereof.
 10. The method ofclaim 3 where greater than 80% of each absorbed target substances isisolated in at most 50 μL volume.
 11. The method according to claim 3wherein the sorbent particles comprise an ion exchange sorbent; areversed phase sorbent; or a normal phase sorbent.
 12. A method ofseparating a target substance from interfering components in a samplemedium as in claim 3 further comprising: the reservoir and well in amulti-well array.
 13. A method of making an extraction devicecomprising: providing a housing having a reservoir with an opening forreceiving fluids and a well having a wall that tapers at an includedangle of between 1 and 30° with an exit spout at a narrow end of thewell and an opening at a wide end of the well, the well including alower diameter of an inverted conical frustum that provides a specifiedaspect ratio when a selected volume of sorbent particles are placed inthe well at that lower diameter; placing a smaller spherical filter witha diameter equal to the lower diameter in the well; placing the selectedvolume of sorbent particles in the well; and placing a larger sphericalfilter, with a diameter sufficiently large to position it above a voidvolume above the volume of sorbent particles, in the well.
 14. Themethod of claim 13 wherein after the smaller spherical filter is placedin the well, it is pressed in place.
 15. The method of claim 13 whereinafter the larger spherical filter is placed in the well, it is pressedin place.
 16. The method of claim 13 wherein the volume of sorbentparticles is placed in the well by pouring a slurry containing thevolume of sorbent particles into the well and allowing the liquid toexit through the exit spout.
 17. The method of claim 13 wherein thefilters are placed in the well by dropping them into the well andallowing them to find a seating point.
 18. A method of making anextraction device comprising: providing a housing having a reservoirwith an opening for receiving fluids and a well having a wall thattapers at an included angle of between 1 and 30° with an exit spout at anarrow end of the well and an opening at a wide end of the well, thewell including the lower diameter of an inverted conical frustum thatprovides a specified aspect ratio when a selected volume of sorbentparticles are placed in the well at that lower diameter; press sealing aspherical filter with a diameter equal to the lower diameter in thewell; placing the volume of sorbent particles in the well; and presssealing a second spherical filter, with a diameter sufficiently large toposition it above a void volume above the volume of sorbent particles,in the well.